The role of ubiquitin in protein degradation was discovered and the main enzymatic reactions of this system elucidated in biochemical studies in a cell-free system from reticulocytes. In this system, proteins are targeted for degradation by covalent ligation to ubiquitin, a 76-amino-acid-residue protein. Briefly, ubiquitin-protein ligation requires the sequential action of three enzymes. The C-terminal Gly residue of ubiquitin is activated in an ATP-requiring step by a specific activating enzyme, E1 (Step 1). This step consists of an intermediate formation of ubiquitin adenylate, with the release of PPi, followed by the binding of ubiquitin to a Cys residue of E1 in a thiolester linkage, with the release of AMP. Activated ubiquitin is next transferred to an active site Cys residue of a ubiquitin-carrier protein, E2 (Step 2). In the third step catalyzed by a ubiquitin-protein ligase or E3 enzyme, ubiquitin is linked by its C-terminus in an amide isopeptide linkage to an -amino group of the substrate protein's Lys residues (Step 3).
Proteins ligated to polyubiquitin chains are usually degraded by the 26S proteasome complex that requires ATP hydrolysis for its action. The 26S proteasome is formed by an ATP-dependent assembly of a 20S proteasome, a complex that contains the protease catalytic sites, with 19S “cap” or regulatory complexes. The 19S complexes contain several ATPase subunits and other subunits that are presumably involved in the specific action of the 26S proteasome on ubiquitinylated proteins. The roles of ATP in the assembly of the 26S proteasome complex and in its proteolytic action are not understood. The action of the 26S proteasome presumably generates several types of products: free peptides, short peptides still linked to ubiquitin via their Lys residues, and polyubiquitin chains (Step 4). The latter two products are converted to free and reusable ubiquitin by the action of ubiquitin-C-terminal hydrolases or isopeptidases (Steps 5 and 6). Some isopeptidases may also disassemble certain ubiquitin-protein conjugates (Step 7) and thus prevent their proteolysis by the 26S proteasome. The latter type of isopeptidase action may have a correction function to salvage incorrectly ubiquitinylated proteins or may have a regulatory role. Short peptides formed by the above processes can be further degraded to free amino acids by cytosolic peptidases (Step 8).
Ubiquitin-mediated degradation of protein is involved in various biological processes. The selective and programmed degradation of cell-cycle regulatory proteins, such as cyclins, inhibitors of cyclin-dependent kinases, and anaphase inhibitors are essential events in cell-cycle progression. Cell growth and proliferation are further controlled by ubiquitin-mediated degradation of tumor suppressors, protooncogenes, and components of signal transduction systems. The rapid degradation of numerous transcriptional regulators is involved in a variety of signal transduction processes and responses to environmental cues. The ubiquitin system is clearly involved in endocytosis and down-regulation of receptors and transporters, as well as in the degradation of resident or abnormal proteins in the endoplasmic reticulum. There are strong indications for roles of the ubiquitin system in development and apoptosis, although the target proteins involved in these cases have not been identified. Dysfunction in several ubiquitin-mediated processes causes pathological conditions, including malignant transformation.
Our knowledge of different signals in proteins that mark them for ubiquitinylation is also limited. Recent reports indicate that many proteins are targeted for degradation by phosphorylation. It was observed previously that many rapidly degraded proteins contain PEST elements, regions enriched in Pro, Glu, Ser, and Thr residues. More recently, it was pointed out that PEST elements are rich in S/TP sequences, which are minimum consensus phosphorylation sites for Cdks and some other protein kinases. Indeed, it now appears that in several (though certainly not all) instances, PEST elements contain phosphorylation sites necessary for degradation. Thus multiple phosphorylations within PEST elements are required for the ubiquitinylation and degradation of the yeast G1 cyclins Cln3 and Cln2, as well as the Gcn4 transcriptional activator. Other proteins, such as the mammalian G1 regulators cyclin E and cyclin D1, are targeted for ubiquitinylation by phosphorylation at specific, single sites. In the case of the IkBα inhibitor of the NF-kB transcriptional regulator, phosphorylation at two specific sites, Ser-32 and Ser-36, is required for ubiquitin ligation. β-cateinin, which is targeted for ubiquitin-mediated degradation by phosphorylation, has a sequence motif similar to that of IkBα around these phosphorylation sites. However, the homology in phosphorylation patterns of these two proteins is not complete, because phosphorylation of other sites of β-catenin is also required for its degradation. Other proteins targeted for degradation by phosphorylation include the Cdk inhibitor Sic1p and the STAT1 transcription factor. Though different patterns of phosphorylation target different proteins for degradation, a common feature appears to be that the initial regulatory event is carried out by a protein kinase, while the role of a ubiquitin ligase would be to recognize the phosphorylated form of the protein substrate. It further appears that different ubiquitin ligases recognize different phosphorylation patterns as well as additional motifs in the various protein substrates.
However, the identity of such E3s is unknown, except for some PULC-type ubiquitin ligases that act on some phosphorylated cell-cycle regulators in the budding yeast. The multiplicity of signals that target proteins for ubiquitin-mediated degradation (and of ligases that have to recognize such signals) is underscored by observations that the phosphorylation of some proteins actually prevents their degradation. Thus the phosphorylation of the c-Mos protooncogene on Ser3 and the multiple phosphorylations of c-Fos and c-Jun protooncogenes at multiple sites by MAP kinases suppress their ubiquitinylation and degradation.
In addition to the families of enzymes involved in conjugation of ubiquitin, a very large family of deubiquitinating enzymes has recently been identified from various organisms. These enzymes have several possible functions. First, they may have peptidase activity and cleave the products of ubiquitin genes. Ubiquitin is encoded by two distinct classes of genes. One is a polyubiquitin gene, which encodes a linear polymer of ubiquitins linked through peptide bonds between the C-terminal Gly and N-terminal Met of contiguous ubiquitin molecules. Each copy of ubiquitin must be released by precise cleavage of the peptide bond between Gly-76-Met-1 of successive ubiquitin moieties. The other class of ubiquitin genes encodes ubiquitin C-terminal extension proteins, which are peptide bond fusions between the C-terminal Gly of ubiquitin and N-terminal Met of the extension protein. To date, the extensions described are ribosomal proteins consisting of 52 or 76–80 amino acids. These ubiquitin fusion proteins are processed to yield ubiquitin and the corresponding C-terminal extension proteins. Second, deubiquitinating enzymes may have isopeptidase activities. When a target protein is degraded, deubiquitinating enzymes can cleave the polyubiquitin chain from the target protein or its remnants. The polyubiquitin chain must also be disassembled by deubiquitinating enzymes during or after proteolysis by the 26 S proteasome, regenerating free monomeric ubiquitin. In this way, deubiquitinating enzymes can facilitate the ability of the 26 S proteasome to degrade ubiquitinated proteins. Third, deubiquitinating enzymes may hydrolyze ester, thiolester, and amide linkages to the carboxyl group of Gly-76 of ubiquitin. Such nonfunctional linkages may arise from reactions between small intracellular compounds such as glutathione and the E1-, E2-, or E3-ubiquitin thiolester intermediates. Fourth, deubiquitinating enzymes may compete with the conjugating system by removing ubiquitin from protein substrates, thereby rescuing them from degradation or any other function mediated by ubiquitination. Thus generation of ubiquitin by deubiquitinating enzymes from the linear polyubiquitin and ubiquitin fusion proteins and from the branched polyubiquitin ligated to proteins should be essential for maintaining a sufficient pool of free ubiquitin. Many deubiquitinating enzymes exist, suggesting that these deubiquitinating enzymes recognize distinct substrates and are therefore involved in specific cellular processes. Although there is recent evidence to support such specificity of these deubiquitinating enzymes, the structure-function relationships of these enzymes remain poorly studied.
Deubiquitinating enzymes can be divided broadly on the basis of sequence homology into two classes, the ubiquitin-specific processing protease (UBP or USP, also known as type 2 ubiquitin C-terminal hydrolase (type 2 UCH)) and the UCH, also known as type 1 UCH). UCH (type 1 UCH) enzymes hydrolyze primarily C-terminal esters and amides of ubiquitin but may also cleave ubiquitin gene products and disassemble polyubiquitin chains. They have in common a 210-amino acid catalytic domain, with four highly conserved blocks of sequences that identify these enzymes. They contain two very conserved motifs, the CYS and HIS boxes. Mutagenesis studies revealed that the two boxes play important roles in catalysis. Some UCH enzymes have significant C-terminal extensions. The functions of the C-terminal extensions are still unknown but appear to be involved in proper localization of the enzyme. The active site of these UCH enzymes contains a catalytic triad consisting of cysteine, histidine, and aspartate and utilizes a chemical mechanism similar to that of papain. The crystal structure of one of these, UCH-L3, has been solved at 1.8 Å resolution. The enzyme comprises a central antiparallel β-sheet flanked on both sides by helices. The β-sheet and one of the helices are similar to those observed in the thiol protease cathepsin B. The similarity includes the three amino acid residues that comprise the active site, Cys95, His169, and Asp184. The active site appears to fit the binding of ubiquitin that may anchor also at an additional site. The catalytic site in the free enzyme is masked by two different segments of the molecule that limit nonspecific hydrolysis and must undergo conformational rearrangement after substrate binding.
UBP (type 2 UCH) enzymes are capable of cleaving the ubiquitin gene products and disassembling polyubiquitin chains after hydrolysis. It appears that there is a core region of about 450 amino acids delimited by CYS and HIS boxes. Many of these isoforms have N-terminal extensions and a few have C-terminal extensions. In addition, there are variable sequences in the core region of many of the isoforms. The functions of these divergent sequences remain poorly characterized. Another interesting function of specific UBPs is the regulation of cell proliferation. It was observed that cytokines induced in T-cells specific deubiquitinating enzymes (DUBs), termed DUB-1 and DUB-2. DUB-1 is induced by stimulation of the cytokine receptors for IL-3, IL-5, and GM-CSF, suggesting a role in its induction for the β-common (betac) subunit of the interleukin receptors. Overexpression of a dominant negative mutant of JAK2 inhibits cytokine induction of DUB-1, suggesting that the regulation of the enzyme is part of the cell response to the JAK/STAT signal transduction pathway. Continued expression of DUB-1 arrests cells at G1; therefore, the enzyme appears to regulate cellular growth via control of the G0–G1 transition. The catalytic conserved Cys residue of the enzyme is required for its activity. DUB-2 is induced by IL-2 as an immediate early (IE) gene that is down-regulated shortly after the initiation of stimulation. The function of this enzyme is also obscure. It may stimulate or inhibit the degradation of a critical cell-cycle regulator.
Cytokines, such as interleukin-2 (IL-2), activate intracellular signaling pathways via rapid tyrosine phosphorylation of their receptors, resulting in the activation of many genes involved in cell growth and survival. The deubiquitinating enzyme DUB-2 is induced in response to IL-2 and is expressed in human T-cell lymphotropic virus-I (HTLV-1)-transformed T cells that exhibit constitutive activation of the IL-2 JAK/STAT (signal transducers and activators of transcription) pathway, and when expressed in Ba/F3 cells DUB-2 markedly prolonged IL-2-induced STAT5 phosphorylation. Although DUB-2 does not enhance IL-2-mediated proliferation, when withdrawn from growth factor, cells expressing DUB-2 had sustained STAT5 phosphorylation and enhanced expression of IL-2-induced genes cis and c-myc. DUB-2 expression markedly inhibited apoptosis induced by cytokine withdrawal allowing cells to survive. Therefore, DUB-2 has a role in enhancing signaling through the JAK/STAT pathway, prolonging lymphocyte survival, and, when constitutively expressed, may contribute to the activation of the JAK/STAT pathway observed in some transformed cells. (Migone, T.-S., et al., Blood. 2001;98:1935–1941).
Protein ubiquitination is an important regulator of cytokine-activated signal transduction pathways and hematopoietic cell growth. Protein ubiquitination is controlled by the coordinate action of ubiquitin-conjugating enzymes and deubiquitinating enzymes. Recently a novel family of genes encoding growth-regulatory deubiquitinating enzymes (DUB-1 and DUB-2) has been identified. DUBs are immediate-early genes and are induced rapidly and transiently in response to cytokine stimuli. By means of polymerase chain reaction amplification with degenerate primers for the DUB-2 complementary DNA, 3 murine bacterial artificial chromosome (BAC) clones that contain DUB gene sequences were isolated. One BAC contained a novel DUB gene (DUB-2A) with extensive homology to DUB-2. Like DUB-1 and DUB-2, the DUB-2A gene consists of 2 exons. The predicted DUB-2A protein is highly related to other DUBs throughout the primary amino acid sequence, with a hypervariable region at its C-terminus. In vitro, DUB-2A had functional deubiquitinating activity; mutation of its conserved amino acid residues abolished this activity. The 5′ flanking sequence of the DUB-2A gene has a hematopoietic-specific functional enhancer sequence. It is proposed that there are at least 3 members of the DUB subfamily (DUB-1, DUB-2, and DUB-2A) and that different hematopoietic cytokines induce specific DUB genes, thereby initiating a cytokine-specific growth response. (Baek, K.-H., et al, Blood. 2001;98:636–642).
Protein ubiquitination also serves regulatory functions in the cell that do not involve proteasome-mediated degradation. For example, Hicke and Riezman have recently demonstrated ligand-inducible ubiquitination of the Ste2 receptor in yeast. Ubiquitination of the Ste2 receptor triggers receptor endocytosis and receptor targeting to vacuoles, not proteasomes. Also, Chen et al. have demonstrated that activation of the IB kinase requires a rapid, inducible ubiquitination event. This ubiquitination event is a prerequisite for the specific phosphorylation of IB and does not result in subsequent proteolysis of the kinase complex. The ubiquitination of Ste2 and IB kinase appears reversible, perhaps resulting from the action of a specific deubiquitinating enzyme.
A large superfamily of genes encoding deubiquitinating enzymes, or UBPs, has recently been identified. UBPs are ubiquitin-specific thiol-proteases that cleave either linear ubiquitin precursor proteins or post-translationally modified proteins containing isopeptide ubiquitin conjugates. The large number of UBPs suggests that protein ubiquitination, like protein phosphorylation, is a highly reversible process that is regulated in the cell.
Interestingly, UBPs vary greatly in length and structural complexity, suggesting functional diversity. While there is little amino acid sequence similarity throughout their coding region, sequence comparison reveals two conserved domains. The Cys domain contains a cysteine residue that serves as the active enzymatic nucleophile. The His domain contains a histidine residue that contributes to the enzyme's active site. More recent evidence demonstrates six homology domains contained by all members of the ubp superfamily. Mutagenesis of conserved residues in the Cys and His domains has identified several residues that are essential for UBP activity.
Recently, a growth regulatory deubiquitinating enzyme, DUB-1, that is rapidly induced in response to cytokine receptor stimulation was identified. DUB-1 is specifically induced by the receptors for IL-3, granulocyte macrophage-colony-stimulating factor, and IL-5, suggesting a specific role for the c subunit shared by these receptors. In the process of cloning the DUB-1 gene, a family of related, cross-hybridizing DUB genes was identified. From this, other DUB genes might be induced by different growth factors. Using this approach, an IL-2-inducible DUB enzyme, DUB-2 and closely related DUB-2a were identified. DUB-1 and DUB-2 are more related to each other than to other members of the ubp superfamily and thereby define a novel subfamily of deubiquitinating enzymes.
Hematopoietic-specific, cytokine induced DUBs in murine system have shown to prolong cytokine receptor, see Migone, T. S., et al. (2001). The deubiquitinating enzyme DUB-2 prolongs cytokine-induced signal transducers and activators of transcription activation and suppresses apoptosis following cytokine withdrawal, Blood 98, 1935–41; Zhu, Y., et al., (1997). DUB-2 is a member of a novel family of cytokine-inducible deubiquitinating enzymes, J Biol Chem 272, 51–7 and Zhu, Y., et al., (1996). The murine DUB-1 gene is specifically induced by the betac subunit of interleukin-3 receptor, Mol Cell Biol 16, 4808–17.). These effects are likely due to the deubiquitination of receptors or other signaling intermediates by DUB-1 or DUB-2, murine analogs of hDUBs. Inhibition of hDUBs may achieve downregulation of specific cytokine receptor signaling, thus modulating specific immune responses.
Cytokines regulate cell growth by inducing the expression of specific target genes. A recently identified a cytokine-inducible, immediate-early gene, DUB-1, encodes a deubiquitinating enzyme with growth regulatory activity. In addition, a highly related gene, DUB-2, that is induced by interleukin-2 was identified. The DUB-2 mRNA was induced in T cells as an immediate-early gene and was rapidly down-regulated. Like DUB-1, the DUB-2 protein had deubiquitinating activity in vitro. When a conserved cysteine residue of DUB-2, required for ubiquitin-specific thiol protease activity, was mutated to serine (C60S), deubiquitinating activity was abolished. DUB-1 and DUB-2 proteins are highly related throughout their primary amino acid sequence except for a hypervariable region at their COOH terminus. Moreover, the DUB genes co-localize to a region of mouse chromosome 7, suggesting that they arose by a tandem duplication of an ancestral DUB gene. Additional DUB genes co-localize to this region, suggesting a larger family of cytokine-inducible DUB enzymes. We propose that different cytokines induce specific DUB genes. Each induced DUB enzyme thereby regulates the degradation or the ubiquitination state of an unknown growth regulatory factor, resulting in a cytokine-specific growth response.
On the basis of these structural criteria, additional members of the DUB subfamily can be identified in the GenBank™. The highest degree of homology is in the Cys and His domains. Additionally, this putative human DUB protein contains a Lys domain (amino acids 400–410) and a hypervariable region (amino acids 413–442).
Murine DUB (mDUB) subfamily members differ from other UBPs by functional criteria as well. mDUB subfamily members are cytokine-inducible, immediate-early genes and may therefore play regulatory roles in cellular growth or differentiation. Also, DUB proteins are unstable and are rapidly degraded by ubiquitin-mediated proteolysis shortly after their induction. mDUB reports demonstrate that specific cytokines, such as IL-2 and IL-3, induce specific deubiquitinating enzymes (DUBs). The DUB proteins may modify the ubiquitin-proteolytic pathway and thereby mediate specific cell growth or differentiation signals. These modifications are temporally regulated. The DUB-2 protein, for instance, is rapidly but transiently induced by IL-2. Interference of DUB enzymes with specific isopeptidase inhibitors may block specific cytokine signaling events.
The prior art teaches some partial sequences with homology to DUBs; specifically Human cDNA sequence SEQ ID NO: 17168 in EP1074617-A2; a human protease and protease inhibitor PPIM-4 encoding cDNA; in WO200110903-A2 and human ubiquitin protease 23431 coding sequence in WO200123589-A2.